This project will examine the role of non-coding regulatory RNAs (ncRNAs) and hypothetical proteins of Enterococcus faecalis in adaptation to growth conditions relevant to opportunistic infections. E. faecalis is a leading cause of hospital-acquired infections whose treatment is impeded by the inherent and acquired antibiotic resistance of the organism, and by its propensity to adapt to growth and survival in hostile conditions, and by its ability to transition between commensal and pathogenic lifestyles. We will study the model laboratory strain OG1RF in both an in-vitro biofilm system (CDC biofilm reactor) and in an experimental endocarditis model. We will use a combination of computational searches, and a powerful new genetic screen we have termed Smart TnSeq to identify hypothetical proteins ncRNAs contributing to competitive fitness in these environments, and begin to study their targets and mechanisms of action. The results of this research will include the following: 1) A more comprehensive picture of the role of ncRNA regulation and hypothetical protein function in this organism under growth conditions relevant to its role in hospital infectios will emerge. 2) A workflow for genome-wide functional and mechanistic studies of regulation by E. faecalis ncRNAs and hypothetical proteins will be validated. 3) In the longer term, new drug and vaccine targets may be identified.